RECOMBINANT BCG OVEREXPRESSSING phoP-phoR

ABSTRACT

Provided are a live recombinant  Mycobacterium bovis -BCG strain and a tuberculosis (TB) vaccine or immunogenic composition comprising a nucleic acid capable of overexpression, the nucleic acid encoding PhoP and PhoR proteins. A method for treatment or prophylaxis of a mammal against challenge by  Mycobacterium tuberculosis  or  Mycobacterium bovis  using the strain is also provided.

FIELD OF THE INVENTION

This invention relates to tuberculosis (TB) vaccines. In particular, theinvention provides a recombinant BCG that overexpresses the phoP-phoRtwo-component regulatory system and confers enhanced protection againsttuberculosis.

BACKGROUND OF THE INVENTION

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), ranksalongside HIV/AIDS as a major cause of mortality by infectious diseasesworldwide. In 2014, TB caused 1.5 million deaths and 9.6 million newinfections. The lack of a protective vaccine, the emergence ofdrug-resistant M. tb strains, and the high rate of M. tb/HIV coinfectioncontinue to fuel the TB epidemic. Bacille Calmette-Guérin (BCG) is theonly licenced TB vaccine and although it is effective againstdisseminated forms of TB in children^(1,2), BCG has limited protectionagainst pulmonary TB in adults, the most common and contagious form ofthe disease. Clinical studies have shown variable efficacies rangingfrom 0 to 80%³⁻⁵.

One hypothesis to explain the variable efficacy of BCG concerns theheterogeneity of BCG strains⁶. BCG was derived from a virulent strain ofMycobacterium bovis through in vitro passaging from 1908-1921.Subsequent worldwide distribution and continuous passaging until the1960s resulted in a number of BCG substrains. Genetic differences amongBCG strains including deletions and duplications of genomic regions andsingle nucleotide polymorphisms (SNPs) have been well documented⁷⁻¹².Whether these differences affect BCG effectiveness against TB is amatter of debate^(6,13), and currently there are insufficient data torecommend one particular strain because of the paucity of clinicaltrials directly comparing multiple BCG strains¹⁴.

Current strategies to improve TB vaccines include the development ofsubunit and live attenuated vaccines^(15,16). However, none of thesubunit vaccines have proved to be superior to BCG in animal models. Thelack of protective efficacy of MVA85A, the most advanced subunitvaccine, in BCG-vaccinated infants in a recent clinical trial¹⁷ furtheremphasizes the importance of live vaccine research¹⁸. A number ofapproaches have been explored to develop live vaccines including thegeneration of recombinant BCG and attenuated M. tb strains. Of these,only a few have proven to be superior to BCG in animal models includingthe three live vaccines (rBCG30, VPM1002, and MTBVAC) that have enteredclinical trials¹⁶. rBCG30 is a recombinant BCG-Tice strain thatoverexpresses antigen Ag85B. rBCG30-vaccination of guinea pigs followedby M. tb challenge resulted in a reduction of the bacterial burden by0.5-1.0 log₁₀ and prolonged survival compared to those immunized withthe parental BCG^(19,20). However, this effect was specific to BCG-Ticesince overexpression of Ag85B in BCG-Connaught did not improveprotection²⁰. VPM1002 is a recombinant BCG that expresses listeriolysinof Listeria monocytogenes. The rationale behind this vaccine was thenotion that listeriolysin could facilitate phagosomal escape of BCG intothe cytosol of macrophages, thereby increasing antigen presentation²¹.BALB/c mice vaccinated with VPM1002 showed a reduction in M. tb burdenby 0.5-1.0 log₁₀ compared to the parental strain^(21,22); however, thisimprovement in protection was not observed in the guinea pig model²³. AphoP deletion mutant of M. tb was evaluated as a vaccine candidate withthe reasoning that attenuated M. tb may share more antigens withclinical strains of M. tb than BCG²⁴ . M. tb ΔphoP provided similarprotection as BCG in mice but better protection in guinea pigs againstM. tb challenge^(24,25). To ensure safety, fadD26 was deleted to furtherattenuate the strain (MTBVAC), which showed a comparable safety profileto BCG-Pasteur or BCG-Danish in SCID mice²⁶.

SUMMARY OF THE INVENTION

The present invention provides tuberculosis vaccines comprising arecombinant Mycobacterium strain that overexpresses phoP-phoR, atwo-component regulatory system. The immunogenicity of current BCGvaccine strains is not sufficient to induce the optimal protection inhost against tuberculosis. In contrast, a genetically engineered BCGstrain of present invention that overexpresses phoP-phoR is moreimmunogenic and provides better protection against tuberculosis. Anygenetically engineered Mycobacterium that overproduces phoP-phoR at alevel sufficient to cause a 2-(or more) fold induction of the PhoP-PhoRregulated genes or proteins may be advantageously used in the practiceof this invention. When the recombinant Mycobacterium of the inventionis administered to a mammalian host, the production of IFN-γ by CD4⁺ Tcells is increased in the host, and this recombinant Mycobacteriumprovides protection against tuberculosis.

There have been over a dozen studies comparing the immune responseinduced by different BCG strains in humans^(14,27). However, only two orthree BCG strains were included in the majority of these studies.Differences in study design such as the choice of BCG strains, age atimmunization, and population size have led to inconclusive results¹⁴.Nonetheless, the largest of these studies, which was led by the WHO inthe 1970s, compared 11 BCG strains in children²⁸. BCG-Prague was foundto be an outlier, exhibiting lower tuberculin reactivity than the otherBCG strains, including BCG-Danish, -Pasteur, -Glaxo, -Japan, -Russia,and -Moreau^(28,29). Concern over its low immunogenicity resulted in itsreplacement by BCG-Russia in Czechoslovakia in 1981, after nearly 30years of use³⁰. Reasons for the low tuberculin reactivity of BCG-Pragueremain unknown. However, the inventor found that phoP in BCG-Prague ispseudogene, containing a 1-bp insertion that disrupts the C-terminalDNA-binding domain⁹. This mutation is specific to BCG-Prague since allother BCG strains contain a wild type (WT) phoP. PhoP is a responseregulator of the PhoP-PhoR two-component system and positively regulatesmore than 40 genes in M. tb , including two T cell antigens (Ag85A,PPE18) that have been used to construct subunit vaccines^(16,31). Assuch, the inventor hypothesized that the low immunogenicity ofBCG-Prague is a result of phoP mutation⁹, and that overexpression ofphoP in BCG may provide an effective means to enhance immunogenicity andtherefore protective efficacy. Consistently, the inventor showed inWO2011/130878A1 that complementation of BCG-Prague with WT phoP oroverexpression of phoP in BCG-Japan increased IFN-γ production.

The present invention is based on the inventor's discovery thatoverexpression of phoP-phoR in BCG strain, such as BCG-Japan, enhancedits immunogenicity and protective efficacy, suggesting that this couldbe a generally applicable approach to improve BCG. It was demonstratedin the present invention that vaccination of C57BL/6 mice with therecombinant strain rBCG-Japan/PhoPR induced higher levels of IFN-γproduction by CD4⁺ T cells than that with the parental BCG. Guinea pigsvaccinated with rBCG-Japan/PhoPR were better protected against challengewith Mycobacterium tuberculosis, showing significantly longer survivaltime, reduced bacterial burdens and less severe pathology, as comparedwith animals immunized with the parental BCG. Taken together, therecombinant BCG of present invention that overexpresses phoP-phoR hasbeen confirmed to confer enhanced protection against tuberculosis thancurrent BCG.

An exemplary amino acid sequence of PhoP is presented in FIG. 1A [SEQ IDNO:1] and an exemplary nucleotide sequence encoding the same ispresented in FIG. 1B [SEQ ID NO:2]. An exemplary amino acid sequence ofPhoR is presented in FIG. 1C [SEQ ID NO:3] and an exemplary nucleotidesequence encoding the same is presented in FIG. 1D [SEQ ID NO:4]. Thesesequences represent PhoP and PhoR from BCG-Pasteur, as presented in thegenome sequence available at the Pasteur Institute's Website(http://genodb.pasteur.fr/cgi-bin/WebObjects/GenoList).

The present invention relates to a recombinant Mycobacterium bovis BCG,which overexpresses DNA encoding PhoP [SEQ ID NO:1; SEQ ID NO:2] andPhoR [SEQ ID NO:3; SEQ ID NO:4].

The present invention relates to a recombinant Mycobacterium bovis BCGcomprising a nucleic acid capable of overexpression, the nucleic acidencoding PhoP [SEQ ID NO:1; SEQ ID NO:2] and PhoR [SEQ ID NO:3; SEQ IDNO:4].

In one embodiment, the recombinant Mycobacterium bovis-BCG strain isselected from the group consisting of Mycobacterium bovis-BCG-Russia,Mycobacterium bovis-BCG-Moreau, Mycobacterium bovis-BCG-Japan,Mycobacterium bovis-BCG-Sweden, Mycobacterium bovis-BCG-Birkhaug,Mycobacterium bovis-BCG-Prague, Mycobacterium bovis-BCG-Glaxo,Mycobacterium bovis-BCG-Denmark, Mycobacterium bovis-BCG-Tice,Mycobacterium bovis-BCG-Frappier, Mycobacterium bovis-BCG-Connaught,Mycobacterium bovis-BCG-Phipps, Mycobacterium bovis-BCG-Pasteur, andMycobacterium bovis-BCG-China.

In addition, the recombinant mycobacteria of the invention need not beconfined to strains of BCG. Those of skill in the art will recognizethat other Mycobacterium strains may also be employed includingattenuated strains of M. tb .

In yet another embodiment, the vaccine of the invention may be a subunitor DNA-vaccine. In some embodiments, the vaccine would be delivered vialung pathogens. For example, the DNA sequences coding for PhoP and PhoRcould be harbored within the chromosome or extra chromosomal nucleicacid of a lung pathogen such as attenuated Pseudomonas aeruginosa, orother known attenuated fungi or viruses. Alternatively, the nucleic acidencoding PhoP and PhoR regulon could be delivered by other means knownto those of skill in the art, e.g., via liposomes, adenoviral vectors,etc.

Another aspect of the invention is a pharmaceutical compositioncomprising a live recombinant Mycobacterium bovis-BCG strain comprisinga nucleic acid capable of overexpression, the nucleic acid encoding PhoP[SEQ ID NO:1; SEQ ID NO:2] and PhoR [SEQ ID NO:3; SEQ ID NO:4].

In a further aspect of the invention there is a vaccine or immunogeniccomposition for treatment or prophylaxis of a mammal against challengeby mycobacteria comprising a live recombinant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of overexpression, the nucleicacid encoding PhoP [SEQ ID NO:1; SEQ ID NO:2] and PhoR [SEQ ID NO:3; SEQID NO:4].

Another aspect of this invention relates to a method for treatment orprophylaxis of a mammal against challenge by Mycobacterium tuberculosisor Mycobacterium bovis comprising administering to the mammal a vaccineor immunogenic composition of the instant invention. In one embodimentthe mammal is a cow. In another embodiment the mammal is a human. In yetanother embodiment the vaccine or immunogenic composition isadministered in the presence of an adjuvant.

A further aspect of the invention is a method for the treatment orprophylaxis of a mammal against cancer comprising administering to themammal a vaccine or immunogenic composition of the current invention. Inone embodiment the cancer is bladder cancer. In another embodiment thevaccine or immunogenic composition is administered in the presence of anadjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows PhoP-PhoR sequences. (a) Amino acid sequence of PhoP ofBCG-Pasteur; (b) DNA sequence of phoP of BCG-Pasteur. (c) Amino acidsequence of PhoR of BCG-Pasteur; (d) DNA sequence of phoR ofBCG-Pasteur.

FIG. 2 shows the cloning vector pME.

FIG. 3 shows the constructed expression vectors for phoP and phoP-phoR.

FIG. 4 shows that rBCG-Japan/PhoPR induces more IFN-γ production by CD4⁺T cells than parental BCG.

FIG. 5 shows that rBCG-Japan/PhoPR prolongs the survival of guinea pigsinfected with M. tb.

FIG. 6 shows that rBCG-Japan/PhoPR reduces the lung pathology of guineapigs infected with M. tb.

FIG. 7 shows that rBCG-Japan/PhoPR is safe in SCID mice.

DETAILED DESCRIPTION OF THE INVENTION

Protection against TB requires cell-mediated immunity, which is notfully understood but involves multiple components including CD4⁺ andCD8⁺ T Cells^(16,32,33). BCG induces a T helper cell 1 (Th1) typeresponse, mostly IFN-γ production by CD4⁺ T cells³⁴. Traditionally,immunogenicity of BCG was determined by measuring the tuberculin (PPD,purified protein derivatives of M. tb) sensitivity induced by thevaccine in children who were tuberculin-negative before vaccination³⁰.While its use as a surrogate measure of protection has been questionedin recent years^(35,36), tuberculin reactivity continues to be used asan in vivo assay for cell-mediated immune response and a marker forimmunogenicity^(19,37). Supporting this, there is a strong associationbetween tuberculin reactivity and PPD-specific IFN-γ levels inBCG-vaccinated infants³⁸. Further, tuberculin reactivity and IFN-γproduction were found to be non-redundant, complementary measures ofanti-TB immunity in young people³⁹. Studies in the 1970s found thatBCG-Prague consistently exhibited significantly lower tuberculinreactivity in children and guinea pigs than the other 10 BCG strainstested^(28,29). Concern over its low immunogenicity resulted in itsreplacement by BCG-Russia in Czechoslovakia in 1981, after nearly 30years of use³⁰.

Reasons for the reduced tuberculin reactivity of BCG-Prague are unknown.I hypothesized that the phoP mutation in BCG-Prague discovered in ourprevious study⁹ contributes to its reduced immunogenicity.

To test this hypothesis, we first complemented BCG-Prague with an intactphoP gene and determined its effect on immunogenicity. The WT phoP genefrom BCG-Pasteur was cloned into a multicopy shuttle vector pME andintroduced into BCG-Prague (rBCG-Prague/PhoP). C57BL/6 mice werevaccinated with the recombinant BCG-Prague strains and the production ofPPD-specific IFN-γ was measured by ELISA. Consistently, rBCG-Prague/PhoPinduced higher levels of PPD-specific IFN-γ release in C57BL/6 mice,which was ˜2.4 fold of that in mice immunized with the parental strain(FIG. 4a , p<0.05).

To test if overexpression of phoP can be used as a generally applicablemethod to improve BCG immunogenicity, we constructed a recombinant BCGoverexpressing phoP in another BCG strain, BCG-Japan and generatedrBCG-Japan/PhoP. PhoR is a histidine kinase that senses an unknownextracellular signal and phosphorylates PhoP³¹. Since BCG-Japan has anintact phoP phoR which is con-transcribed in its genome, we alsogenerated rBCG-Japan/PhoPR, overexpressing both phoP and phoR tomaintain the functional ratio of this two-component system.

Consistent with the results obtained with BCG-Prague, bothrBCG-Japan/PhoP and rBCG-Japan/PhoPR induced significantly higher levelsof PPD-specific IFN-γ production in C57BL/6 mice than the parentalstrain (FIG. 4b ). Interestingly, among the three strains,rBCG-Japan/PhoPR induced the highest level of IFN-γ.

To determine the source of IFN-γ induced by the recombinant BCG strains,we also performed intracellular cytokine staining and FACS analyses. Wefound that CD4⁺ T cells were likely responsible for the enhanced IFN-γrelease (FIG. 4c ). The frequency of IFN-γ producing CD4⁺ T cells inmice vaccinated with rBCG-Japan/PhoPR was ˜3.5 fold of that in micevaccinated with the parental strain, which is in agreement with the folddifference (˜2.6 fold) of total IFN-γ production between these twogroups (FIG. 4b, c ).

In contrast, neither recombinant BCG-Japan strain induced a robust CD8⁺T cell response compared to the sham-immunized control. No significantinduction of other cytokines (IL-2, TNF, IL-12, IL-4, IL-5, and IL-10)by the recombinant BCG-Japan strains was detected.

Taken together, these results suggest that the phoP mutation ispartially responsible for the low immunogenicity of BCG-Prague. Moreimportantly, overexpression of phoP-phoR in BCG-Japan further boostedIFN-γ production by CD4⁺ T cells, suggesting that this could be agenerally applicable approach to enhance the protective efficacy of BCG.

To examine if overexpression of phoP-phoR improves BCG-mediatedprotection against M. tb infection, we performed a long-term (10 months)guinea pig survival experiment. Guinea pigs (11 per group) werevaccinated with rBCG-Japan/PhoPR, the parental strain, or PBS and werechallenged aerogenically with 1,000 CFU/lung of M. tb H37Rv 8 weekspost-vaccination. Guinea pigs were euthanized at the humane end-pointand survival curves were plotted using a Kaplan-Meier analysis.

The median survival time for the PBS, the parental, and therBCG-Japan/PhoPR groups were 18, 27, and 39 weeks, respectively (FIG. 5a). Log-rank analysis revealed that the rBCG-Japan/PhoPR group survivedsignificantly longer than the parental BCG group (p<0.05) and the PBSgroup (p<0.0001). The parental group also survived significantly longerthan the PBS group (p<0.01).

Compared to unvaccinated animals, the parental BCG prolonged thesurvival of guinea pigs by 9 weeks, while rBCG-Japan/PhoPR prolonged thesurvival of guinea pigs by 21 weeks (133% improvement over the parentalgroup). At week 43 post-challenge when the experiment was terminated,only one guinea pig in the parental group survived compared to fouranimals in the rBCG-Japan/PhoPR group. All animals in the PBS groupsuccumbed to infection by week 25.

The guinea pig lungs and spleen were further analyzed after the animalswere euthanized at the humane or experimental endpoint. Similar toprevious observation, the rBCG-Japan/PhoPR group had ˜1.7 log₁₀ lower M.tb counts in the lungs than the parental BCG group (FIG. 5b , p<0.05).The M. tb burden in the spleen of the rBCG-Japan/PhoPR group was also˜1.0 log₁₀ lower than the parental BCG group and this difference isapproaching significance (p=0.066). Consistently, the rBCG-Japan/PhoPRgroup had the lowest lung and spleen weights compared to the other twogroups (FIG. 5d, e ).

The lungs of five animals were subjected to histological analysis. Theyinclude one animal from each group that reached the humane endpoint, thesole survivor of the parental group, and one of the four survivors ofthe rBCG-Japan/PhoPR group at week 43. Interestingly, the mortality ofguinea pigs appeared to be associated with the extent of tissue damagein the caudal lobe. The three animals euthanized before the end ofexperiment had extensive (FIG. 6a ) or partial consolidation (FIG. 6b, c) in the caudal lobes in addition to extensive consolidation in thecranial lobes. In contrast, the two survivors appeared to have healthytissues in the caudal lobe, despite the fact that the one from theparental group also had extensive tissue damage in the cranial lobe(FIG. 6d ). Strikingly, the survivor of the rBCG-Japan/PhoPR groupappeared to have normal lungs with no visible lung consolidation ineither lobe (FIG. 6e ).

PhoP is considered a virulence factor of M. tb ²⁴ partially because itpositively regulates the secretion of EsxA, an important effector of M.tb virulence⁴⁰. As such, it is possible that overexpression of phoP-phoRin BCG may increase virulence and compromise safety. On the other hand,since esxA is part the region of difference 1 (RD1) that is absent inthe genomes of all BCG strains¹², the extent to which overexpression ofphoP-phoR contributes to BCG virulence remains unclear.

To address this, we first infected SCID mice with the recombinantBCG-Japan strains and monitored bacterial growth in target organs for upto 6 weeks. Interestingly, there was no significant difference betweenthe growth of rBCG-Japan/PhoPR and the parental BCG in the lungs orspleen during the course of the experiment (FIG. 7a, b ). However,overexpression of phoP alone increased replication of BCG-Japan in SCIDmice compared to both the parental strain and rBCG-Japan/PhoPR.

Next, we performed a long-term SCID mice survival experiment.BCG-Pasteur, which is among the most virulent of the BCG strains⁴¹, wasalso included in this experiment for comparison. The median survivaltime of SCID mice infected with BCG-Pasteur, rBCG-Japan/PhoP, andrBCG-Japan/PhoPR were 7, 14, and 19 weeks, respectively (FIG. 7c ). AllSCID mice infected with the parental strain or PBS survived until week20 when the experiment was terminated. Log-rank analysis revealed thatthe rBCG-Japan/PhoP group had significantly reduced survival compared tothe rBCG-Japan/PhoPR group (p=0.02) and the parental group (p<0.001),which is consistent with the higher bacterial burdens observed in thisgroup in the short-term infection experiment (FIG. 7a, b ). TherBCG-Japan/PhoPR group also showed reduced survival compared to theparental group (p=0.02). Importantly, both rBCG-Japan/PhoPR andrBCG-Japan/PhoP were significantly less virulent than BCG-Pasteur(p<0.001) in SCID mice. Taken together, these results suggest that theoverexpression of phoP-phoR in BCG-Japan does not increase itsreplication in SCID mice and causes only a modest increase in virulence.

Taken together, we demonstrated that recombinant BCG overexpressingphoP-phoR confers enhanced protection against tuberculosis. The superiorprotection of rBCG-Japan/PhoPR was demonstrated in guinea pigs,evidenced by significantly prolonged survival and reduced pathology,which is a hallmark for testing novel vaccines in animal models. Guineapigs are the “gold standard” animal model for testing TB vaccineefficacy'. The pathogenesis of disease, pathological lesions andresponse to BCG vaccination are similar to those described in humans.

There is consensus that novel TB vaccine candidates must meet criteriato advance from the discovery stage into pre-clinical development. Thesecriteria include a robust induction of IFN-γ, better protective efficacythan BCG, and a comparable safety profile to BCG in the establishedanimal models⁴². The rBCG-Japan/PhoPR strain has already satisfied thesecriteria and thus is a promising candidate for future clinicaldevelopment.

M. bovis BCG is also used in the treatment of bladder cancer. Numerousrandomized controlled clinical trials indicate that intravesicaladministration of BCG can prevent or delay tumor recurrence⁴³. Thedetails of how BCG exerts this effect remain to be determined. However,the antitumor response requires an intact T-cell response, and involvesincreased expression of Th1-type cytokines⁴⁴. As such, a BCG strain suchas rBCG-Japan/PhoPR demonstrating increased Th1 cytokine IFN-γ mayprovide enhanced antitumor activity.

In summary, we use recombinant BCG strains that overexpress phoP phoR asvaccines to prevent TB and other mycobacterial infections. Theserecombinant BCG vaccines induce better protective immunity againsttuberculosis.

In the genetically engineered (i.e., recombinant) Mycobacterium of theinvention, the PhoP and PhoR proteins are overexpressed, i.e., these twoproteins are expressed at a level that exceeds that of a suitablecontrol organism, such as the same Mycobacterium that has not beengenetically engineered to overexpress PhoP and PhoR. Those of skill inthe art are well acquainted with comparative measurements of proteinactivity, and with the use of suitable standards and controls for suchmeasurements.

The overexpression of PhoP and PhoR proteins in a Mycobacterium may becarried out by any suitable method known in the art. Generally, themethod will involve linking nucleic acid sequences encoding the PhoP andPhoR proteins to expression control sequences that are not, in nature,linked to phoP and phoR genes. Those of skill in the art will recognizethat many such expression-control sequences are known and would besuitable for use in the invention. For example, if constitutiveexpression of phoP and phoR genes is desired, expression-controlsequences (e.g., promoters and associated sequences) include but are notlimited to: Mycobacterium optimal promoter: hsp65, ace or msp 12promoter, T7 promoter, etc. Alternatively, overexpression of phoP andphoR may be inducible for example, under the tetracycline induciblepromoters.

The proteins, polypeptides or peptides encoded according to theinvention include naturally-occurring proteins, polypeptides orpeptides, or the homologs thereof which have the same function asnaturally-occurring proteins, polypeptides or peptides. Such homologsinclude proteins, polypeptides or peptides having at least 60%,preferably about 70% or more, 80% or more, and most preferably 90% ormore, e.g., 95%, 96%, 97%, 98 or 99% homology to the amino acidsequences of the naturally-occurring proteins, polypeptides or peptides,e.g., to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:3.Such homologs include proteins, polypeptides or peptides withsubstitution, addition and deletion of one or more (e.g., 1-50, 1-20,1-10, 1-5) amino acid residues in the amino acid sequences of thenaturally-occurring proteins, polypeptides or peptides (e.g., in theamino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:3). Suchhomologs include especially proteins, polypeptides or peptides withconserved amino acid substitution(s).

The term “PhoP” and “PhoR”, as used therein, refer to the two-componentregulatory system PhoP-PhoR. PhoP is a response regulator and PhoR is ahistidine kinase. PhoP or PhoR encoded according to the inventionincludes naturally-occurring, functional PhoPs and PhoRs, e.g., fromgenus Mycobacterium, preferably from Mycobacterium tuberculosis, orMycobacterium bovis, or the homologs thereof as described above.Exemplary amino acid sequences of PhoP and PhoR are presented in FIG. 1aSEQ ID NO:1 and FIG. 1c SEQ ID NO:3.

The term “overexpress”, “overexpressing” or “overexpression”, as usedtherein, refers to the protein level of a target gene expressed in arecombinant bacterium is higher than the level of the same proteinexpressed in the initial bacterium which is not recombinant. Forexample, the protein of interest expressed in a recombinant bacterium is1.1, 1.5, 2, 4, 10, 20, 50, 100, 500, 1000 or more fold than anon-recombinant bacterium. The overexpression can be carried out bygenetic engineering such as the use of a multiple copy plasmids and/orthe use of strong promoters.”

Variations of Nucleic Acid Molecules Modifications

Many modifications may be made to the nucleic acid molecule DNAsequences disclosed in this application and these will be apparent toone skilled in the art. The invention includes nucleotide modificationsof the sequences disclosed in this application (or fragments thereof)that encode proteins or peptides in bacterial or mammalian cells whichhave the same function as the proteins or peptides disclosed in thisapplication. Modifications include substitution, insertion or deletionof one or more (e.g., 1-50, 1-20, 1-10, 1-5) nucleotides or altering therelative positions or order of one or more (e.g., 1-50, 1-20, 1-10, 1-5)nucleotides.

Nucleic acid molecules may encode conservative amino acid changes inPhoP and PhoR proteins. The invention includes functionally equivalentnucleic acid molecules that encode conservative amino acid changes andproduce silent amino acid changes in PhoP and PhoR proteins. Methods foridentifying empirically conserved amino acid substitution groups arewell known in the art (see for example, Wu, Thomas D. “DiscoveringEmperically Conserved Amino Acid Substitution Groups in Databases ofProtein Families ”(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8877523&dopt=Abstract).

Nucleic acid molecules may encode non-conservative amino acidsubstitutions, additions or deletions in phoP and/or phoR gene. Theinvention includes functionally equivalent nucleic acid molecules thatmake non-conservative amino acid changes within the amino acid sequencesin PhoP and PhoR proteins. Functionally equivalent nucleic acidmolecules include DNA and RNA that encode peptides, peptides andproteins having non-conservative amino acid substitutions (preferablysubstitution of a chemically similar amino acid), additions, ordeletions but which also retain the same or similar function to the PhoPand PhoR proteins or peptides disclosed in this application. Theinvention includes the DNAs or RNAs encoding fragments or variants ofPhoP and PhoR protein.

The fragments are useful as immunogens and in immunogenic compositions.

PhoP and PhoR like-activity of such fragments and variants is identifiedby assays as described below.

Sequence Identity

The nucleic acid molecules of the invention also include nucleic acidmolecules (or a fragment thereof) having at least about: 60% identity,at least 70% identity, at least 80% identity, at least 90% identity, atleast 95% identity, at least 96% identity, at least 97% identity, atleast 98% identity or, most preferred, at least 99% or 99.5% identity toa nucleic acid molecule of the invention and which are capable ofexpression of nucleic acid molecules in bacterial or mammalian cells.Identity refers to the similarity of two nucleotide sequences that arealigned so that the highest order match is obtained. Identity iscalculated according to methods known in the art. For example, if anucleotide sequence (called “Sequence A”) has 90% identity to a portionof SEQ ID NO: 2, then Sequence A will be identical to the referencedportion of SEQ ID NO: 2 except that Sequence A may include up to 10point mutations (such as substitutions with other nucleotides) per each100 nucleotides of the referenced portion of SEQ ID NO: 2.

Sequence identity (each construct preferably without a coding nucleicacid molecule insert) is preferably set at least about: 70% identity, atleast 80% identity, at least 90% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity or,most preferred, at least 99% or 99.5% identity to the sequences providedin SEQ ID NO: 2 or its complementary sequence). Sequence identity willpreferably be calculated with the GCG program from Bioinformatics(University of Wisconsin). Other programs are also available tocalculate sequence identity, such as the Clustal W program (preferablyusing default parameters; Thompson, J D et al., Nucleic Acid Res.22:4673-4680), BLAST P, BLAST X algorithms, Mycobacterium avium BLASTNat The Institute for Genomic Research (http:tigrblast.tigr.org/),Mycobacterium bovis, M. bovis BCG (Pastuer), M. marinum, M. leprae, M.tuberculosis BLASTN at the Wellcome Trust Sanger Institute(http://www.sanger.ac.uk/Projects/Microbes/), M. tuberculosis BLASTsearches at Institute Pasterur (Tuberculist)(http://genolist.pasteur.fr/TubercuList/), M. leprae BLAST searches atInstitute Pasteur (Leproma) (http://genolist.pasteur.fr/Leproma/), M.paratuberculosis BLASTN at Microbial Genome Project, University ofMinnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ andhttp://www.cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mptbhome.html),various BLAST searches at the National Center for BiotechnologyInformation—USA (http://www.ncbi.nlm.nih.gov/BLAST/) and various BLASTsearches at GenomeNet (Bioinformatics Center—Institute for ChemicalResearch) (http://blast.genome.ad.jp/).

Since the genetic code is degenerate, the nucleic acid sequences in SEQID NO:2 and SEQ ID NO:4 are not the only sequences which may code for apolypeptide having PhoP and PhoR activities. This invention includesnucleic acid molecules that have the same essential genetic informationas the nucleic acid molecules described in SEQ ID NO:2 and SEQ ID NO:4.Nucleic acid molecules (including RNA) having one or more nucleic acidchanges compared to the sequences described in this application andwhich result in production of the polypeptides shown in SEQ ID NO:1 andSEQ ID NO:3 are within the scope of the invention.

Other functional equivalent forms of PhoP and PhoR proteins-encodingnucleic acids can be isolated using conventional DNA-DNA or DNA-RNAhybridization techniques.

Hybridization

The invention includes DNA that has a sequence with sufficient identityto a nucleic acid molecule described in this application to hybridizeunder stringent hybridization conditions (hybridization techniques arewell known in the art). The present invention also includes nucleic acidmolecules that hybridize to one or more of the sequences in [SEQ IDNO:2] and SEQ ID NO:4 or their complementary sequences. Such nucleicacid molecules preferably hybridize under high stringency conditions(see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most RecentEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).High stringency washes have preferably low salt (preferably about 0.2%SSC) and a temperature of about 50-65° C.

Vaccines

One skilled in the art knows the preparation of live recombinantvaccines. Typically, such vaccines are prepared as injectable, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the protein encapsulated inliposomes. The live immunogenic ingredients are often mixed withexcipients that are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants that enhance the effectiveness of the vaccine. Examplesof adjuvants which may be effective include but are not limited to:aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to asMTP-PE), and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80™ emulsion.

The effectiveness of an adjuvant may be determined by measuring theamount of antibodies directed against an immunogenic polypeptidecontaining a Mycobacterium tuberculosis antigenic sequence resultingfrom administration of the live recombinant Mycobacterium bovis-BCGvaccines that are also comprised of the various adjuvants.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or reinforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgment of thepractitioner.

In addition, the live recombinant Mycobacterium bovis-BCG vaccineadministered in conjunction with other immuneregulatory agents, forexample, immune globulins.

A subject of the present invention is also a multivalent vaccine formulacomprising, as a mixture or to be mixed, a live recombinantMycobacterium bovis-BCG vaccine as defined above with another vaccine,and in particular another recombinant live recombinant Mycobacteriumbovis-BCG vaccine as defined above, these vaccines comprising differentinserted sequences.

Pharmaceutical Compositions

The pharmaceutical compositions of this invention are used for thetreatment or prophylaxis of a mammal against challenge by Mycobacteriumtuberculosis or Mycobacterium bovis. The pharmaceutical compositions ofthis invention are also used to treat patients having degenerativediseases, disorders or abnormal physical states such as cancer.

The pharmaceutical compositions can be administered to humans or animalsby methods such as tablets, aerosol administration, intratrachealinstillation and intravenous injection.

EXAMPLES Example 1: Cloning Vector pME

A kanamycin resistant shuttle vector which contains a T7 promoter wasobtained as follows. The pDrive cloning vector (obtained from Qiagen)was cut with EcoRI and self-ligated to generate pDRI. The pDRI wasdigested with SspI and the 1903 bp fragment product was isolated. ThepMD31 shuttle vector (Wu et al., 1993, Molecular Microbiology, 7,407-417) was digested with SspI and the 3379 bp fragment was isolated.The two SspI generated fragments were ligated to generate pME (5282 bp),which contains the original T7 promoter of the pDRIVE. The cloningvector pME is shown in FIG. 2.

Example 2: Construction of Expression Vectors for phoP and phoP-phoR

An 1028 bp product containing phoP as well as the 257 bp upstream regionof the phoP translational start site was amplified by PCR using aforward primer phoP-F (5′-AAAAAGGTACCGCTTGTTTGGCCATGTCAAC-3′) and areverse primer phoP-R (5′-AAAAACTGCAGGCTGCCGATCCGATTAACTAC-3′) whichcontain a KpnI and a PstI restriction site (underlined), respectively.Using these restriction sites, the PCR product was ligated to a shuttlevector pME (pME-PhoP). Similarly, a vector which expresses the phoP-phoRoperon (pME-PhoPR) was constructed by cloning a 2501 bp PCRproduct—containing phoP and phoR (as well as the intergenic regionbetween the two genes, 177 bp upstream of the phoP start codon, and 78bp downstream of phoR stop codon)—into pME. This product was amplifiedby PCR from BCG-Pasteur genomic DNA using forward primer phoPR-F(5′-AAAAAGGTACCGGTCGCAATACCCACGAG-3′) and reverse primer phoPR-R(5′-AAAAACTGCAGCCTCAGTGATTTCGGCTTTG-3′) containing a KpnI and a PstIsite (underlined), respectively. The constructs were confirmed by DNAsequencing.

The constructs as well as the empty vector pME were electroporatedseparately into BCG-Prague and BCG-Japan. Generation of a recombinantBCG was accomplished as follows: Cells of M. bovis BCG-Japan (ATCC35737) and M. bovis BCG-Prague (ATCC 35742) were transformed withplasmid pME-PhoP or pME-PhoPR by electroporation, and electroporatedcells were plated onto 7H11 media plates supplemented with 10% OADC(Difco) and 25 μg/ml of kanamycin. After 4 weeks of incubation at 37°C., individual colonies were selected and grown in 7H9 liquid mediasupplemented with 10% ADC (Difco) plus 25 μg/ml of kanamycin. Afterculturing for 3 weeks in this media, plasmid DNA from the selectedclones was isolated and confirmed by DNA sequencing. Frozen stocks ofrecombinant BCG strains were subsequently made by mixing 1 ml of culturewith 1 ml of 50% sterile glycerol solution and stored at −80° C.

The constructed expression vectors for phoP and phoP-phoR are shown inFIG. 3.

Example 3: rBCG-Japan/PhoPR Induces More IFN-γ Production by CD4⁺ TCells than Parental BCG

Female C57BL/6 mice were purchased from Charles River Laboratories andwere age-matched (6 weeks) within each experiment. Four mice per groupwere inoculated subcutaneously on the scruff of the neck withapproximately 5×10⁴ CFU in 0.2 ml PBS/0.01% Tween 80 of BCG strains.Control mice were given 0.2 mL of PBS/0.01% Tween 80. After 8 weeks,mice were euthanized, splenocytes were isolated. Quantitativemeasurements of IFN-γ production was determined using an enzyme-linkedimmunosorbant assay (ELISA) using the OptEIA Mouse IFN-γ ELISA set (BDBiosciences). Samples used for ELISAs were supernatants from splenocytesstimulated by PPD (10 μg/ml) for 72 hours. Briefly, samples were addedin triplicate to 96-well, flat-bottomed plates (Nunc MaxiSorp)pre-coated with capture antibody and measurements were carried outfollowing the manufacturer's protocol. The absorbance was read at 450 nmon a microplate reader (TECAN infinite M200). IFN-γ levels werecalculated based on a standard curve generated using an IFN-γ standard.The results are shown in FIGS. 4a and 4b . Data are shown as mean±SEM,after subtraction of reading from samples without PPD stimulation. InFIG. 4a , the two-tailed unpaired student t-test was performed; in FIG.4b , One-way ANOVA and Bonferroni multiple comparison tests wereperformed. *p<0.05, **p<0.01, ***p<0.001.

To determine the source of IFN-γ induced by the recombinant BCG strains,we also performed intracellular cytokine staining and FACS analyses.Briefly, splenocytes were seeded at 2×10⁶ cells/well in 100 μl intriplicate and stimulated with 2.5 μg/well of purified proteinderivative (PPD) (Statens Serum Institute, Denmark) or complete RPMI(cRPMI; RPMI/10% FBS/1% L-glutamine/1% penicillin/streptomycin) as acontrol and incubated at 37° C. and 5% CO₂. After 19 hours ofstimulation, GolgiPlug (BD Biosciences) was added in a 1:1000 finaldilution and incubated for an additional 5 hours. After a total of 24hours stimulation, plates were centrifuged at 1400 rpm for 5 minutes at4° C. The supernatant was removed and the cell pellet was washed in 200μl FACS Buffer (0.5% BSA/PBS), and resuspended in Fc Block(eBiosciences) diluted in FACS Buffer (1:400) and incubated for 15minutes on ice in the dark. An additional 150 μl of FACS Buffer wasadded, mixed, and plates were centrifuged at 1400 rpm for 5 minutes at4° C. Supernatant was removed and cells were stained for extracellular Tcell surface markers: CD3-PE, CD4-FITC, and CD8a-PercyPCy5.5 (BDBiosciences) diluted in FACS Buffer, and incubated for 30 minutes on icein the dark. Following extracellular marker staining, the cells werewashed with 150 μl FACS Buffer and permeabilized and fixed with 1×CytoFix/CytoPerm (BD Biosciences) for 20 minutes. Cells were then washedwith 1× PermWash (BD Biosciences) and incubated with IFN-γ-APC (BDBiosciences) for 30 minutes to stain for intracellular IFN-γ. Cells werecentrifuged as above, resuspended in 200 μl FACS Buffer, and analyzed ona BD FACSCalibur™ flow cytometer (BD Biosciences). A total of 300 000events per sample were collected in the lymphocyte gate and analyzedusing FlowJo V7.6. Gates for analysis were set based on isotypecontrols. Data are shown as mean±SEM, after subtraction of reading fromsamples without PPD stimulation. The result is shown in FIG. 4c , andone-way ANOVA and Bonferroni multiple comparison tests were performedfor statistical analysis. *p<0.05, **p<0.01, ***p<0.001.

Example 4: rBCG-Japan/PhoPR Prolongs the Survival of Guinea PiesInfected with M. tb

Groups of 11 female Hartely guinea pigs (200-250 g) were purchased fromCharles River Laboratories, and they were vaccinated subcutaneously with5×10⁴ CFU of BCG-Japan containing pME (rBCG-Japan/pME), pME-PhoPR(rBCG-Japan/PhoPR) in 0.2 ml PBS/0.01% Tween80 or PBS/0.01% Tween80alone as a control. At eight weeks post-vaccination, guinea pigs werechallenged with 1,000 CFU of M. tb H37Rv by an aerosol route using aGlasCol nebulizer. Guinea pigs were euthanized at the humane end-pointand survival curves were plotted using the Kaplan-Meier method. Theresult is shown in FIG. 5a . Log-rank test was performed to compare eachpair of the survival curves. *p<0.05, **p<0.01, ***p<0.0001.

The lungs and spleen of guinea pigs euthanized at the humane end-pointwere harvested. The spleen and the entire right lung were homogenizedseparately and plated on 7H11 agar to quantify the M. tb burden in eachorgan. Colonies were counted after incubation at 37° C. for three weeks.The results are shown in FIGS. 5b and 5c . The organ weights of allanimals in each group were measured and the results are shown in FIGS.5d and 5e . Data in FIGS. 5b to 5e are shown as mean±SEM, and One-wayANOVA and Bonferroni multiple comparison tests were performed. *p<0.05,**p<0.01, ***p<0.001.

Example 5: rBCG-Japan/PhoPR Reduces the Lung Pathology of Guinea PiesInfected with M. tb

Six guinea pigs from the experiment described in FIG. 4 were chosen forhistology analysis. These include one guinea pig from each group thatreached the humane endpoint at weeks 25, 27, and 39, respectively (FIGS.6a to 6c ). The pME (FIG. 6b ) and PhoPR (FIG. 6c ) guinea pigsrepresent the sixth animal to be euthanized from each group (median).The sole survivor of the pME group (FIG. 6d ) and one randomly chosensurvivor of the PhoPR group (FIG. 6e ) were included. For each guineapig, sections of caudal and cranial lobes of the left lungs wereanalyzed by hematoxylin-eosin staining. Briefly, formalin-fixed tissueswere embedded into paraffin blocks. Serial sections (5 μm thick) wereprepared and they went through deparafinization process with threechanges of xylene (3 minutes each) before being rehydrated with fourwashes of alcohol (100%, 100%, 95%, 70%, 3 minutes each). Sections werestained with hematoxylin and eosin (EMD Chemicals) and examined usingCytation™ 5 (BioTek).

Example 6: rBCG-Japan/PhoPR is Safe in SCID Mice

Short-term bacterial burden assay: Female Fox Chase CB17 SCID mice(Charles River Laboratories) were age-matched (7 weeks old). Groups of12 mice were infected intravenously via a lateral tail vein with 10⁵ CFUof rBCG-Japan/pME, rBCG-Japan/ PhoP, or rBCG-Japan/PhoPR in 0.2 mL ofPBS/0.01% Tween80 or PBS/0.01% Tween80 alone as a control. At weeks 1,3, and 6 post-infection, mice (n=4 at each time point) were euthanizedand the lungs and spleen were harvested, homogenized in PBS, and platedon 7H11 agar to determine bacterial burden in the lungs (FIG. 7a ) andspleen (FIG. 7b ). Data are showed as mean±SD. Two-Way ANOVA andBonferroni multiple comparison tests were performed. *p<0.05, **p<0.01,***p<0.001.

Long term survival assay: Groups of ten age-matched female SCID mice (7weeks old) from Charles River Laboratories were infected intravenouslyvia a lateral tail vein with 10⁷ CFU of BCG-Pasteur, threeaforementioned recombinant BCG-Japan strains or PBS/0.01% Tween80 asdescribed above. Three mice from each group were euthanized at day 1post-infection (for assessing infection dose) while the remaining micewere monitored weekly until they reached a humane endpoint (loss of 20%maximal body weight). Survival curves were plotted using theKaplan-Meier method. The results are shown in FIG. 7c . Log-rank testwas performed to compare each pair of the survival curves. *p<0.05,***p<0.001.

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1. A live recombinant Mycobacterium bovis-BCG strain comprising nucleicacids capable of overexpression, the nucleic acids encoding PhoP andPhoR proteins.
 2. The live recombinant Mycobacterium bovis-BCG strain ofclaim 1, wherein the PhoP and PhoR proteins are from Mycobacteriumtuberculosis or Mycobacterium bovis.
 3. The live recombinantMycobacterium bovis-BCG strain of claim 1, wherein the nucleic acidsencode (i) the amino acid sequence shown in SEQ ID NO: 1; or (ii) a PhoPprotein with the substitution, addition, or deletion of one or moreamino acids in the amino acid sequence shown in SEQ ID NO: 1; or p1(iii) the amino acid sequence shown in SEQ ID NO: 3; or (iv) a PhoRprotein with the substitution, addition, or deletion of one or moreamino acids in the amino acid sequence shown in SEQ ID NO:
 3. 4. Thelive recombinant Mycobacterium bovis-BCG strain of claim 1, wherein thenucleic acids comprise (i) the nucleotide sequence shown in SEQ ID NO:2; or (ii) a sequence that hybridizes to the nucleotide sequence of (i)under a stringent hybridization condition; or (iii) a sequence having atleast 60% sequence identity to the nucleotide sequence shown in SEQ IDNO: 2; or (iv) the nucleotide sequence shown in SEQ ID NO: 4; or (v) asequence that hybridizes to the nucleotide sequence of (iv) under astringent hybridization condition; or (vi) a sequence having at least60% sequence identity to the nucleotide sequence shown in SEQ ID NO: 4.5. The live recombinant Mycobacterium bovis-BCG strain of claim 1wherein the nucleic acid molecule has undergone modification.
 6. Thelive recombinant Mycobacterium bovis-BCG strain of claim 1 wherein theMycobacterium bovis-BCG strain is selected from Mycobacteriumbovis-BCG-Russia (ATCC number: 35740), Mycobacterium bovis-BCG-Moreau(ATCC number: 35736), Mycobacterium bovis-BCG-Japan (ATCC number:35737), Mycobacterium bovis-BCG-Sweden (ATCC number: 35732),Mycobacterium bovis-BCG-Birkhaug (ATCC number: 35731), Mycobacteriumbovis-BCG-Prague (ATCC number: 35742), Mycobacterium bovis-BCG-Glaxo(ATCC number: 35741), Mycobacterium bovis-BCG-Denmark (ATCC number:35733), Mycobacterium bovis-BCG-Tice (ATCC numbers: 35743, 27289),Mycobacterium bovis-BCG-Frappier (ATCC: 35746, SM-R; ATCC: 35747,INH-R), Mycobacterium bovis-BCG-Connaught (ATCC: 35745) , Mycobacteriumbovis-BCG-Phipps (ATCC number: 35744), Mycobacterium bovis-BCG-Pasteur(ATCC number: 35734), BCG-Mexican (ATCC number: 35738) and Mycobacteriumbovis-BCG-China (Shanghai Institute of Biological Product).
 7. Apharmaceutical composition comprising the live recombinant Mycobacteriumbovis-BCG strain of claim
 1. 8. A vaccine or immunogenic composition fortreatment or prophylaxis of a mammal against challenge by mycobacteriacomprising the live recombinant Mycobacterium bovis-BCG strain ofclaim
 1. 9. The vaccine or immunogenic composition of claim 8 whereinthe mycobacteria is Mycobacterium tuberculosis or Mycobacterium bovis.10. The vaccine or immunogenic composition of claim 7 further comprisinga pharmaceutically acceptable carrier.
 11. The vaccine or immunogeniccomposition of claim 7 further comprising an adjuvant.
 12. The vaccineor immunogenic composition of claim 8 further comprising immunogenicmaterials from one or more other pathogens.
 13. A method for treatmentor prophylaxis of a mammal against challenge by Mycobacteriumtuberculosis or Mycobacterium bovis comprising administering to themammal the live recombinant Mycobacterium bovis-BCG strain of claim 1.14. The method of claim 13 wherein the mammal is a cow.
 15. The methodof claim 13 wherein the mammal is a human.
 16. The method of claim 13wherein the live recombinant Mycobacterium bovis-BCG strain isadministered in the presence of an adjuvant.
 17. A method for treatmentor prophylaxis of a mammal against cancer comprising administering tothe mammal the live recombinant Mycobacterium bovis-BCG strain ofclaim
 1. 18. The method of claim 17 wherein the live recombinantMycobacterium bovis-BCG strain is administered in the presence of anadjuvant.
 19. The method of claim 17 wherein the cancer is bladdercancer.